During the past four years, we have used mutant mammalian cells to clone three new genes essential to the SREBP pathway that regulate cholesterol and fatty acid metabolism in mammalian cells. These three genes are SREBP cleavage-activating protein (SCAP), Site-1 protease (S1P), and Site-2 protease (S2P). The mutant cells have also proven crucial to our analysis of the function of these loci. We will continue to use the tools of mammalian cell genetics to pursue the hypothesis that additional essential genes regulating the SREBP signaling pathway remain to be discovered. In particular, we seek to identify the gene encoding a putative "ER retention protein" that is required for the sterol-regulated movement of the SCAP/SREBP complex between the endoplasmic reticulum (ER) and the Golgi. This protein may hold the key to the cholesterol feedback phenomenon. In addition, we will initiate a new direction, in Drosophila, to extend our genetic analysis of the SREBP pathway to an organism that cannot synthesize cholesterol. In cultured Drosophila cells, our aim is to identify metabolites that regulate SREBP activity in flies, as well as to identify the gene targets of this pathway. The lack of cholesterol synthesis in flies will allow us to study the regulation of SREBP signaling by non-sterol metabolites in the absence of the complications of the sterol-mediated regulation observed in mammalian systems. We will study the SREBP pathway in vivo by creating mutant flies lacking SREBP, S1P, S2P, and SCAP. These mutants will enable us to identify and characterize phenotypes associated with the loss of function of the SREBP pathway in an animal in which cholesterol feedback is not a normal control mechanism. Once such phenotypes are known, we will use genetic strategies unique to Drosophila to screen for genes involved in the SREBP pathway that would otherwise be difficult to identify in the mammalian system.